Both chloride-binding sites are required for KCC2-mediated transport

The K+–Cl− cotransporter 2 (KCC2) plays an important role in inhibitory neurotransmission, and its impairment is associated with neurological and psychiatric disorders, including epilepsy, schizophrenia, and autism. Although KCCs transport K+ and Cl− in a 1:1 stoichiometry, two Cl− coordination sites were indicated via cryo-EM. In a comprehensive analysis, we analyzed the consequences of point mutations of residues coordinating Cl− in Cl1 and Cl2. Individual mutations of residues in Cl1 and Cl2 reduce or abolish KCC2WT function, indicating a crucial role of both Cl− coordination sites for KCC2 function. Structural changes in the extracellular loop 2 by inserting a 3xHA tag switches the K+ coordination site to another position. To investigate, whether the extension of the extracellular loop 2 with the 3xHA tag also affects the coordination of the two Cl− coordination sites, we carried out the analogous experiments for both Cl− coordinating sites in the KCC2HA construct. These analyses showed that most of the individual mutation of residues in Cl1 and Cl2 in the KCC2HA construct reduces or abolishes KCC2 function, indicating that the coordination of Cl− remains at the same position. However, the coupling of K+ and Cl− in Cl1 is still apparent in the KCC2HA construct, indicating a mutual dependence of both ions. In addition, the coordination residue Tyr569 in Cl2 shifted in KCC2HA. Thus, conformational changes in the extracellular domain affect K+ and Cl−-binding sites. However, the effect on the Cl−-binding sites is subtler.

Within the CCC family, the KCC2 is of particular interest.It is the major Cl − extrusion transporter in most adult inhibitory neurons of the central nervous system where it lowers [Cl − ] i (9,22,41).Its action enables fast hyperpolarizing postsynaptic inhibition because of Cl − influx mediated by the binding of the inhibitory neurotransmitters gamma-aminobutyric acid and glycine to their ionic gamma-aminobutyric acid type A and glycine receptors (9,41).Mice with disruption of the gene SLC12A5, which encodes both KCC2 splice variants (KCC2a and KCC2b), die shortly after birth because of motor deficits (42,43).Dysfunction of KCC2 is associated with several neurological and psychiatric disorders, including epilepsy, neuropathic pain, spasticity, ischemic insults, brain trauma, schizophrenia, and autism (44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54).These severe consequences raised pharmacological interest in the generation of small-molecule KCC2 potentiators that enhance its activity (55)(56)(57).This requires a thorough understanding of the structure and functioning of KCC2.
None of these Cl − -binding sites have been functionally investigated in KCC2, and more importantly, it is still not known whether both sites are required for transport.Therefore, we performed an extensive functional analysis of residues coordinating Cl − in Cl 1 and Cl 2 sites.Our analyses revealed that both Cl − coordinating sites are required for KCC2 function.
Recently, we showed that a 3xHA tag in KCC2 EL2 connecting TM3 and TM4 (KCC2 HA ) results in subtle conformational changes of the ECD and affects K + ion binding (59).This conformational change likely switches the classical K + -binding site to an alternative K + -binding site consisting of Pro 409 and Ser 410 in TM6 and Tyr 446 in TM7 (Fig. 2B) (59).Here, we used the KCC2 HA construct to analyze, whether the extension of the EL2 also leads to a shift of the Cl 1 and Cl 2 sites.Our data show that this conformational change affects ion coordination of both K + and both Cl − , indicating a functional coupling of these sites.

Structural requirements for KCC2 transport activity
Cl − -binding sites in KCC2b HA Previously, we showed that an mmKCC2b variant with an insertion of a 3xHA tag in EL2 results in subtle conformational changes of ECD and switches the K + -binding site to an alternative K + -binding site predicted to consist of Pro 409 , Ser 410 (in TM6), and Tyr 446 (in TM10) (59).To investigate, whether this extension of EL2 also has an impact on the two Cl − -binding sites, we here generated the following seven 3) was generated using ClustalW in Geneious.The TMs, K + , and Cl − -binding sites were annotated according to the cryo-EM-based three-dimensional structure of mmKCC2 and hsNKCC1 (20).Cl − -binding sites in Cl 1 located in TM1 (G, V, and I) are marked with red dots.Cl − -binding sites in Cl 2 located in TM6 (G, I, and M) and TM10 (Y) are marked with blue dots.K + -binding sites located in TM1 (N and I), TM3 (Y), and TM6 (P and T) are marked with a blue asterisk.Alternative K + sites located in TM6 (P and S) and TM7 (Y) are marked with a red asterisk.The insertion of the HA tag of KCC2b HA at the end of EL2 between TM3 and TM4 is highlighted in red.HA, hemagglutinin; hs, Homo sapiens; KCC, K + -Cl − cotransporter; mm, Mus musculus; TM, transmembrane helix.-binding sites impairs KCC2b activity.HEK-293 cells were transiently transfected with rnKCC2b WT or rnKCC2 variants with mutations in the chloride-binding site 1.Cells were then seeded in parallel for Tl + flux measurements and immunocytochemistry.A, Tl + flux measurements were performed to determine the transport activity.The suggested Cl 1 residue mutants in TM1 rnKCC2b G113A (48% ± 10%, p = 2.11 × 10 −09 compared with rnKCC2b WT ), rnKCC2b V114T (87% ± 17%, p = 0.0009 compared with rnKCC2b WT ), and KCC2b I115E (46% ± 11%, p = 5.65 × 10 −19 compared with rnKCC2b) showed diminished KCC2 activity (Table 1).The graph represents the data of at least five independent measurements including three technical replicates per independent measurement, normalized to rnKCC2b WT .Statistical analysis can be seen in Table 1 ( 2 -binding site impairs KCC2b activity.HEK-293 cells were transiently transfected with rnKCC2b WT or rnKCC2b variants with mutations in the chloride-binding site 2. Cells were then seeded in parallel for Tl + flux measurements and immunocytochemistry.A, Tl + flux measurements were performed to determine the transport activity.The Tl + flux determines abolished transport activity for rnKCC2b G413A in TM6 (41% ± 17%, p = 0.13 compared with mock).rnKCC2b I414E (49% ± 9%, p = 2.38 × 10 −9 compared with rnKCC2b WT ) and rnKCC2b M415Q (52% ± 9%, p = 5.75 × 10 −14 compared with rnKCC2b WT ) in TM6 and rnKCC2b Y569F (71% ± 18%, p = 8.04 × 10 −8 compared with rnKCC2b WT ) in TM10 diminished KCC2 transport activity (100% ± 16%) (Table 2).The graph represents the data of at least five independent measurements including three technical replicates per independent measurement, normalized to rnKCC2b WT .Statistical analysis can be seen in Table 2 (***p < 0.001, two-sample t test with Benjamini-Hochberg adjustment for multiple comparisons).B, mutants: mmKCC2b HA-G113A , mmKCC2b HA-V114T , mmKCC2b HA-I115E for Cl 1 and mmKCC2b HA-G413A , mmKCC2b HA-I414E , mmKCC2b HA-M415Q , and mmKCC2b HA- Y569F for Cl 2 .The mmKCC2b HA construct advantageously displays the same transport activity as rnKCC2b WT and thus enables a systematic investigation of ion-binding sites via mutagenesis (59).
To test how equivalent mutations in rnKCC2b affect its transport activity, we generated the mutations: rnKCC2b P409H , rnKCC2b S410A , and rnKCC2b Y446F in rnKCC2b WT .All mutants showed similar transfection rates in HEK-293 compared with rnKCC2b WT (Fig. 8B).HEK-293 cells transiently expressing KCC2b displayed a significantly higher KCC2 transport activity (100% ± 16%, p = 2.23 × 10 −30 ) than mock-transfected control cells (34% ± 12%, p = 2.23 × 10 −30 ; Fig. 8A, Table 6).The constructs rnKCC2b P409H (48% ± 11%, p = 2.39 × 10 −14 compared with KCC2b WT ) and rnKCC2b S410A (69% ± 11%, p = 2.15 × 10 −6 compared with KCC2b WT ) showed diminished transport activities, whereas rnKCC2b Y446F (188% ± 59%, p = 5.93 × 10 −6 compared with rnKCC2b WT ) enhanced KCC2 activity (Fig. 8A, Table 6).Thus, Pro 409 likely is important for coordinating K + at the "normal" K + -binding site as well as at the alternative K + site.We previously showed that mutation of Pro 432 to histidine in mmKCC2a diminished KCC2 transport activity (59).This is in line with our results here and underpin its general role in K + coordination.The  immunocytochemistry was used to monitor the transfection rate of the rnKCC2 variants (green) and cell staining by DAPI (blue).Representative immunocytochemical images were used for the biological replicates.Since the readings for mock and KCC2 wt of all Tl + -based activity measurements were combined, the representative figure for mock and KCC2 wt was used here as it is also available in Figure 3 HEK-293 cells were transiently transfected with mmKCC2b HA or mmKCC2b HA variants with mutations in the chloride-binding site 1.Cells were then seeded in parallel for Tl + flux measurements and immunocytochemistry.A, Tl + flux measurements were performed to determine the transport activity.The Tl + flux determines for mmKCC2b HA-G113A (44% ± %, p = 0.026 compared with mock) and mmKCC2 HA-I115E (47% ± 13%, p = 0.02 compared with mock) in TM1 show abolished transport activity.mmKCC2b HA-V114T (64% ± 13%, p = 1.91 × 10 −18 compared with mmKCC2b HA ) showed diminished transport activity compared with KCC2b HA (100% ± 11%).The graph represents the data of at least five independent measurements including three technical replicates per independent measurement, normalized to mmKCC2b HA .Statistical analysis can be reduced activity of rnKCC2b S410A may be explained by a weak coordinating role of Ser 410 at Cl 1 .Mutations of Ser 430 in KCC1 and Ser 495 in KCC3 to alanine have already been shown to reduce KCC activity (29).The only residue that showed a strikingly completely different property is Tyr 446 .In mmKCC2b HA , mutation of Tyr 446 to phenylalanine abolished KCC2 activity, whereas Tyr 446Phe in rnKCC2b enhanced KCC2 activity.This confirms that Tyr 446 plays a crucial role in coordinating K + at the alternative K + site.To fully ensure that the insertion of the HA tag leads to a shift of the K + coordination site here, a cryo-EM structure of KCC2 HA is required.
Mutation of Tyr in TM10 that coordinates binding of Cl − in Cl 2 reduces or abolishes the activity of KCC1, KCC3, KCC4, NKCC1, and NCC indicating that the Cl 2 site is important for their function (27)(28)(29)35).Here, we unambiguously demonstrated that mutation of residues coordinating Cl − in Cl 1 (Gly 113 , Val 114 , and Ile 115 in TM1) or Cl 2 (Gly 413 , Ile 414 , Met 415 in TM6 and Tyr 569 in TM10) significantly reduced or abolished KCC2 activity.In addition, Tyr 195 in TM3 has a bifunctional role in stabilizing K + and Cl − in Cl 1 (20).Mutation of Tyr 195 reduced KCC2 activity indicating a crucial role in coordinating both ions (59).Analogous experiments in KCC1 (Tyr 216 ), KCC3 (Tyr 283 ), or KCC4 (Tyr 216 ) support this hypothesis (27)(28)(29).Thus, both suggested that Cl −-binding sites are important for KCC2 function. Thi impairment of the transport activity likely results from intrinsic conformational changes. However we cannot exclude a contribution of decreased cell surface abundance of KCC2 to impaired transport activity but consider this unlikely.
The requirement of both Cl −-binding sites and their evolutionary conservation in KCCs is enigmatic since they transport K + and Cl − in a 1:1 ratio.One explanation is that one binding site is important for K + -coupled Cl − transport and the other Cl − site "allosterically" affects the binding of the other ions (23,29,58).Since the K + and the Cl 1 -binding sites are within 3.4 Å and thus in close proximity, it was hypothesized that ions coordinate with each other to transport both ions in a stoichiometric ratio (Fig. 2A) (29,58).In contrast, the center of Cl 2 is 7.4 Å away from the coordination center of the K + ion.Therefore, a direct coupling of K + and Cl − in Cl 2 is not given here (Fig. 2A).In this scenario, the Cl 2 site has an "allosteric" effect in stabilizing the coordination of the K + -binding site (29,58).In addition, the binding of Cl − at Cl 2 may strengthen the interactions between TM6a+b and TM10 to couple conformational changes during ion translocation (29,58).This scenario is supported by MD simulations in KCC1 (29).Another scenario suggested that Cl 2 presents the transporting site (28).Considering that there is only one universal Cl −-binding site in CCCs, it was assumed that this is not the Cl 1 site, since NCC does not transport K + , and therefore, coupling the transport of K + and Cl − at Cl 1 is not necessary (28).However, the recent cryo-EM structure of NCC showed that NCC has two Na + and two Cl − coordinating sites, which ensure a ratio of 1:1 (35).It is therefore possible that KCCs also have a second previously undetected K + site, which then ensures a 1:1 ratio.
KCCs mainly serve as K + and Cl − extruders.Yet, under nonphysiological conditions (high extracellular [K + ]), they can change transport direction to load cells with K + and Cl − (60).It is hence also possible that both Cl − sites can act as a transporting or "allosteric" site depending on the transport direction.This aspect is supported by the fact that an asymmetry of ion binding in outward-or inward-mediated ion transport was observed (58,61).In the physiological outward-mediated transport direction, intracellular ions bind randomly (58,61), likely because of the close proximity of K + and Cl − at the Cl 1 site.Binding of Cl − to Cl 2 then induces a conformational change required for ion translocation.In contrast, in the nonphysiological inward-mediated transport direction, extracellular ions bind in a determined order (Cl − → K + ) (58,61).Here, Cl − might bind first at Cl 2 before binding of K + and Cl − at the Cl 1 site.Binding of Cl − at site 1 (allosteric site) may then induce a conformation that facilitates ion translocation.In our present analyses, we investigated the inward-mediated transport of Tl + (congener of K + ) and Cl − .A, Tl + flux measurements were performed to determine the transport activity.The Tl + flux measurements showed that mmKCC2b HA-G413A (31% ± 6%, p = 0.09 compared with mock) and mmKCC2b HA-I414E (43% ± 19%, p = 0.09 compared with mock) abolished KCC2 transport activity.Substitution of mmKCC2b HA-M415Q diminished KCC2 transport activity (67% ± 18%, p = 8.35 × 10 −8 compared with mmKCC2b HA ).Contrary, mmKCC2b HA-Y569F in TM10 (91% ± 22%, p = 0.08 compared with mmKCC2b HA ) showed no significant impairment of transport activity compared with KCC2b HA (100% ± 11%).The graph represents the data of at least five independent measurements including three technical replicates per independent measurement, normalized to mmKCC2b HA .Statistical analysis can be seen in Table 4 (***p < 0.001, two-sample t test with Benjamini-Hochberg adjustment for multiple comparisons).
To take a closer look to the function of both Cl − sites, we used a modified KCC2 construct, in which the EL2 is extended by a 37-amino acid-long HA tag.This modification, which importantly does not compromise transport activity, results in subtle conformational changes of ECD, which is transduced to TM1 and TM6 (59).These changes most probably alter K +binding sites in KCC2 HA (59).Because of this conformational change, the K + -binding site switches to an alternative K +binding site consisting of Pro 409 and Ser 410 in TM6 and Tyr 446 in TM7 instead of Asn 110 , Tyr 195 (TM1), and Thr 412 (TM3) (Fig. 2, A and B) (59).K + therefore moves toward Tyr 446 , which is then 3.5 Å far away from Tyr 446 and thus is able to stabilize the K + together with the other coordination sites (Fig. 2B).Mutation of these alternative residues in the KCC2 HA variant abolished KCC2 activity, which corroborates the presence of an alternative K + -binding site in KCC2 HA .
In order to find out whether the extension of the EL2 with the 3xHA tag also affects the coordination of the two Cl − coordination sites, we carried out the analogous experiments for both Cl − coordinating sites in the KCC2 HA construct.
Residues that coordinate Cl − in the Cl 1 site are in close proximity to the K + -binding site.Thus, both ions are coupled with each other with a 3.4 Å distance between them (Fig. 2A).The extension of EL2 with the 3xHA tag shifts the K + coordination site from Tyr 195 in TM3 in KCC2 WT to Tyr 446 in TM7 in KCC2 HA (59).However, residues coordinating Cl − at Cl 1 in KCC2 HA remain at the suggested Cl − coordination positions.Although the K + coordination sites shifted and the Cl 1 coordinating sites remain at the same position, both ions are in close proximity and the distance between them is 2.9 Å (Fig. 2B).Since K + and the alternative K + site are both in close proximity to Cl − in Cl 1 , coupling of both ions is present in both KCC2 WT and KCC2 HA .Interestingly, in the K + -independent NCC, mutation of Tyr 386 to alanine (analogous to Tyr in KCC2 [TM7]) abolishes its function (35).In NCC, Tyr 386 coordinates Cl − at the Cl 1 site through a water molecule (35).Tyr 386 also interacts with Ile 150 and Ser 350 from the broken helices of TM1 and TM6 to mediate conformational coupling during ion coordination (35).This underlines a central role of Tyr 446 in coordination of K + and Cl − in KCC2 HA and likely in the coupling mechanism.Thus, subtle conformational changes in the ECD directly affect K + coordination and slightly Cl − coordination in Cl 1 , wherein a coupling of both ions is still given.Undoubtedly, it is of great interest to analyze the structure of KCC2 HA by MD simulations, but unfortunately, no experimental data for the structure of KCC2 HA are presently available.Moreover, the MD simulations by Zhang et al. (24) on the available cryo-EM structure of KCC2 WT were performed over 1 μs, which would be a publication on its own legitimacy and therefore beyond the scope of this study.
Similar to KCC2b, mutations of Gly 413 , Ile 414 , and Met 415 in TM6 of KCC2 HA significantly reduced or abolished transport, indicating that main-chain interactions of these residues are important for Cl − binding.In contrast, a difference exists for Tyr 569 in TM10 between both constructs, as its mutation reduced transport activity in KCC2b but not in KCC2 HA .The most likely scenario is that the HA taginduced conformational changes that are transduced to TM1 and TM6 result in changes in the spatial localization of TM6 relative to TM10.Whereas residues in TM6 still coordinate Cl − , the residue coordinating Cl − in TM10 switches to an alternative position.This results overall in a slightly altered Cl 2 coordination site.
In summary, our data showed that both Cl − coordination sites are important for KCC2 transport activity.Furthermore, the conformational changes in the ECD by interactions with other extracellular loops affect the coordination of both K + and Cl − .Note, however, that the effect on the Cl − -binding sites is subtler.

Experimental procedures
Graphical representation of K + and Cl − coordination sites Structural figures have been prepared using CCP4mg (62).Manual alternation of the K + -binding site in the PDB file (7D99) has been performed with Coot (63) after careful visual inspection of the electron density map.The alternative K +binding site would be suitable to accommodate this ion without any obvious clashes and a minimum distance of 2.5 Å to the nearest atom in the surrounding.

Construction of the expression clones
Site-directed mutagenesis of rat KCC2b (NM_134363.1) and mouse KCC2b (NM_020333.2) with an HA tag in the extracellular loop 2 3xHA tag (IFYPYDVPDYAGYPYDVPDYA GSYPYDVPDYAAHAAA) was performed according to the QuikChange mutagenesis system (Stratagene) (16,64,65).Representative immunocytochemical images were used for the biological replicates.Since the readings for mock of all Tl + -based activity measurements were combined, the representative figure for mock and KCC2 HA was used here as it is also available in Figure 3 (for mock) and Structural requirements for KCC2 transport activity A B Figure 7. Mutations of the alternative K + -binding site in KCC2b HA abolishes KCC2 activity.HEK-293 cells were transiently transfected with mmKCC2b HA or mmKCC2b HA variants with mutations in the alternative K + .Cells were then seeded in parallel for Tl + flux measurements and immunocytochemistry.A, Tl + flux measurements were performed to determine the transport activity.The constructs mmKCC2b HA-P409H (30% ± 13%, p = 0.22 compared with mock), mmKCC2b HA-S410A (33% ± 10%, p = 0.44 compared with mock), and mmKCC2b HA-Y446F (39% ± 10%, p = 0.44 compared with mock) abolished transport activity (100% ± 11%).The graph represents the data of at least five independent measurements including three technical replicates per independent

Structural requirements for KCC2 transport activity
Forward oligonucleotides for the generation of the mutations are given in Tables 7 and 8.All generated clones were verified by sequencing (LGC Genomics).

Cell culturing
For immunocytochemistry and measurement of K + -Cl − cotransporter activity, HEK-293 cells were transiently transfected with the respective constructs, using Turbofect (Fermentas).Cells were seeded in a 6-well plate 24 h prior to transfection.The Dulbecco's modified Eagle's medium was replaced 4 h before transfection.About 150 μl Opti-MEM (Invitrogen), 6 μl Turbofect, and the corresponding DNA amount were thoroughly mixed and incubated for 20 min at room temperature (RT).The mixture was applied to the cells that were then shaken at 300 rpm for 10 min at RT.
For K + -Cl − cotransporter activity measurements, transfected HEK-293 cells were plated at a concentration of 1 × 10 5 cells/well in a 0.1 mg/ml poly-L-lysine-coated black 96-well culture dish (Greiner Bio-One 24 h after transfection.Each transfected construct was plated out three times and represents three technical replicas.For the generation of independent biological replicates, the constructs were transfected separately.The remaining cells were plated on 0.1 mg/ml poly-L-lysine-coated glass coverslips.After 18 h, coverslips were proceeded for immunocytochemical analysis to determine transfection rates, which were routinely between 20 and 30%.

Immunocytochemistry
For immunocytochemistry, all steps were performed at RT. HEK-293 cells grown on poly-L-lysine-coated coverslips were fixated for 10 min with 4% paraformaldehyde in 0.2 M phosphate buffer.Afterward, the cells were washed three times with PBS before the blocking solution (2% bovine serum albumin and 10% goat serum in PBS) was applied for 30 min.Primary antibody solution (anti-KCC2 N1-12; 1:1000 dilution; Neuromab) was added in carrier solution (0.3% Triton X-100, 1% bovine serum albumin, and 1% goat serum in PBS) and incubated for 1 h.After washing three times with PBS, the secondary antibody, which was conjugated to a fluorescent probe (Alexa Flour 488 goat antimouse; 1:1000dilution; Thermo Fisher Scientific), was added to the carrier solution and incubated for 1 h.Again, the cells were washed three times with PBS and completely dried.The dried coverslips were mounted onto glass slides with Mowiol (Roth) and 4 0 ,6-diamidine-2phenylindole (1:1000 dilution; Roth).Photomicrographs were taken using an Olympus fluorescence microscope (Olympus BX63).

Determination of the K + -Cl − cotransport activity
Transport activity of KCC2 was determined by Cl −dependent uptake of Tl + in HEK-293 cells as described previously (14,59,66).To initiate the flux measurement, the medium in the 96-well culture dish was replaced by 80 μl hypotonic preincubation buffer (100 mM N-methyl-D-glucamine-chloride, 5 mM Hepes, 5 mM KCl, 2 mM CaCl 2 , 0.8 mM MgSO 4 , 5 mM glucose, pH 7.4; osmolarity: 175 mmol/kg ± 2) with 2 μM FluoZin-2 AM dye (Invitrogen) plus 0.2% (w/v) Pluronic F-127 (Invitrogen) and incubated for 48 min at RT. Afterward, cells were washed three times with 80 μl preincubation buffer and incubated for 15 min with 80 ml preincubation buffer including 0.1 mM ouabain to block the activity of the Na + /K + ATPase.Then, the 96-well plate was placed into a fluorometer (Fluoroskan FL), and each well was injected with 40 μl 5× Thallium stimulation buffer (12 mM Tl 2 SO 4 , 100 mM N-methyl-D-glucamine, 5 mM Hepes, 2 mM KCl, 2 mM CaCl 2 , 0.8 mM MgSO 4 , 5 mM glucose, pH 7.4).The fluorescence was measured in a kinetic-dependent manner (excitation 485 nm, emission 538 nm, one frame in 6 s in a 200-speriod) across the entire cell population in a single well.By using linear regression of the initial values of the slope of Tl + -stimulated fluorescence increase, the transport activity was calculated.The absolute values are normalized by setting the slope of the KCC2 WT or KCC2 HA construct as 100% and calculating the percentage of the activity of the mutants respectively.Normalization is needed to subtract naturally occurring fluctuations of the fluorescence increase.A comparison of the absolute versus normalized values for exemplar mutations can be seen in Figs.S1-S3.

Statistical analysis
The normalized transport activities of the respective mutant were tested against control samples (rnKCC2b WT , measurement, normalized to mmKCC2b HA .Statistical analysis can be seen in Table 5 (***p < 0.001, two-sample t test with Benjamini-Hochberg adjustment for multiple comparisons).B, immunocytochemistry was used to monitor the transfection rate of the mmKCC2 HA variants (green) and cell staining by DAPI (blue).Representative immunocytochemical images were used for the biological replicates.Since the readings for mock of all Tl + -based activity measurements were combined, the representative figure for mock and KCC2 HA was used here as it is also available in Figure 3 (for mock) and Structural requirements for KCC2 transport activity mmKCC2b HA , and mock), using a two-sample t test after Student's t test for similar variances between samples.In exceptional cases, where the standard deviation differs by more than a factor of 2, Welch's t test was used (67).To avoid pseudoreplication, the number of degrees of freedom was deflated according to the size of independent preparations each with three technical replicates.The false discovery rate was controlled, and p values were corrected using the Benjamini-Hochberg method (68).The chance of false-positive results (type 1 errors) was reduced by choosing p values <0.01.A Tukey honestly significant difference test after Tukey and Kramer was used for multiple comparison for the analyses in supporting information.

Figure 2 .BFigure 3 .
Figure 2. Structural homology model of KCC2 based on PDB entry 7D99.A, structural overview of the KCC4 dimer (PDB code: 7D99), the closest homolog of mmKCC2, colored by its homodimeric subunits in yellow and blue.Cl − ions are presented as green spheres, and the K + ion is shown as a purple sphere.Right side upper, zoom into the K + coordination site.Right side middle, close-up of the first Cl − ion-binding site (Cl 1 ).Right side down, atoms of residues participating in the coordination are indicated by dashed lines.B, structural comparison between the K + -binding site as observed in 7D99 (left) and the suggested alternative binding site (right).Shown are residues that coordinate the K + ion.The residue annotation is according to the rnKCC2b construct.KCC, K + -Cl − cotransporter; mm, Mus musculus; PDB, Protein Data Bank; rn, Rattus norvegicus.

BFigure 8 .
Figure 8. Mutations of the alternative K + binding in KCC2b differentially affect KCC2 function.HEK-293 cells were transiently transfected with rnKCC2b WT or rnKCC2b variants with mutations in the alternative K + .Cells were then seeded in parallel for Tl + flux measurements and immunocytochemistry.A, Tl + flux measurements were performed to determine the transport activity.The constructs rnKCC2b P409H (48% ± 11%, p = 2.39 × 10 −14 compared with rnKCC2b WT ) and rnKCC2b S410A (69% ± 11%, p = 2.15 × 10 −6 compared with rnKCC2b WT ) diminished KCC2 transport activity, whereas rnKCC2b Y446F (188% ± 59%, p = 5.93 × 10 −6 compared with rnKCC2b WT ) enhanced KCC2 activity.The graph represents the data of at least five independent B, immunocytochemistry was used to monitor the transfection rate of the rnKCC2 variants (green) and cell staining by DAPI (blue).Representative immunocytochemical images were used for the biological replicates.Since the readings for mock and KCC2 wt of all Tl + -based activity measurements were combined, the representative figure for mock and KCC2 wt was used here as it is also available in Figure3.The scale bar represents 200 μm.DAPI, 4 0 ,6-diamidine-2-phenylindole; HEK-293, human embryonic kidney 293 cell line; KCC, K + -Cl − cotransporter; Rn, Rattus norvegicus; TM, transmembrane helix.

Table 1
Transport activity of Cl 1 -binding site mutations in KCC2b

Table 2
Transport activity of Cl 2 -binding site mutations in KCC2b

Table 3
Transport activity of Cl 1 -binding site mutations in KCC2b HA

Table 4
Transport activity of Cl 2 -binding site mutations in KCC2b HA

Table 5
Transport activity of alternative K + -binding site mutations in KCC2b HA

Table 6
Transport activity of alternative K + -binding site mutations in KCC2b

Table 7
Forward primers used for site-directed mutagenesis in rnKCC2b and mmKCC2b of the chloride-binding sites measurements including three technical replicates per independent measurement, normalized to rnKCC2b WT .Statistical analysis can be seen in Table6(***p < 0.001, two-sample t test with Benjamini-Hochberg adjustment for multiple comparisons).

Table 8
Forward primers used for site-directed mutagenesis in rnKCC2b and mmKCC2b of the alternative K + -binding site